IntroductionInfertility is a major global public health concern, affecting approximately 15–20% of couples of reproductive age worldwide1. According to the World Health Organization (WHO), infertility is defined as the inability to conceive after one year of regular, unprotected intercourse before the age of 352. Both male and female factors contribute equally to infertility, and its etiology is multifactorial, involving congenital abnormalities, hormonal imbalances, lifestyle habits, environmental exposures, and psychological stress3. Among these, urogenital infections caused by bacteria, viruses, fungi, protozoa, and mycoplasmas are increasingly recognized as important but often overlooked contributors, particularly in cases of unexplained infertility4.Mycoplasma hominis and Mycoplasma genitalium are among the most prevalent sexually transmitted pathogens associated with infertility in both men and women5. Members of the class Mollicutes and family Mycoplasmataceae, these organisms are the smallest self-replicating prokaryotes, characterized by the absence of a cell wall. They can colonize the urogenital tract as commensals or opportunistic pathogens, often persisting asymptomatically. Persistent colonization can lead to chronic reproductive tract infections such as cervicitis, endometritis, salpingitis, and prostatitis, which may impair fertility and affect the outcomes of assisted reproductive technologies, including in vitro fertilization (IVF)6.The prevalence of Mycoplasma infections varies by demographic characteristics, sexual behavior, and diagnostic approaches, with higher rates reported among younger, sexually active populations7. Treatment generally involves antibiotics such as tetracyclines, macrolides, aminoglycosides, and fluoroquinolones, as β-lactam antibiotics are ineffective due to the absence of a cell wall8. However, increasing antimicrobial resistance poses a significant therapeutic challenge. Mutations in the 23S rRNA gene (A2058G, A2059G) confer resistance to macrolides, while mutations in the parC gene (S83, D87) are associated with fluoroquinolone resistance9,10. Molecular detection and sequencing of these mutations are now considered essential tools for monitoring resistance and guiding effective antimicrobial therapy11.In Iran—and particularly in Hormozgan Province, Bandar Abbas—comprehensive data on the prevalence, antimicrobial susceptibility, and molecular characteristics of genital Mycoplasma species remain extremely limited. To our knowledge, no prior study in this region has simultaneously examined both infertile and fertile men and women using an integrated molecular diagnostic framework. Addressing this gap, the present study investigates the prevalence of M. hominis and M. genitalium among these individuals, evaluates their antimicrobial susceptibility to erythromycin, azithromycin, and moxifloxacin through the microbroth dilution method, and characterizes parC and 23S rRNA resistance-associated mutations in M. genitalium via Sanger sequencing. Although this work does not capture the full spectrum of resistance mechanisms, it specifically targets clinically relevant mutations with the greatest impact on therapeutic efficacy. By combining sex-specific molecular detection, targeted susceptibility testing, and focused mutational analysis, this study provides novel, region-specific insights into the epidemiology, clinical significance, and potential treatment challenges of genital Mycoplasma infections in southern Iran, thereby addressing a critical knowledge gap and informing future surveillance and management strategies.Methods and materialsSubjectsThis descriptive cross-sectional study was conducted during the first half of 2024 at the Ome Leila Infertility Center in Bandar Abbas, Hormozgan Province, Iran. A total of 400 participants were enrolled during a single study period and clinically assigned to two groups: infertile individuals (n = 200) and fertile controls (n = 200).Fertility status was determined by specialist physicians, including gynecologists for female participants and andrologists for male participants, based on clinical evaluation. Infertile participants were defined as individuals with a confirmed clinical diagnosis of infertility. Fertile controls were defined as individuals with evidence of fertility, including a history of at least one live birth and/or a normal fertility evaluation, as assessed by the treating physician.The fertile control group was recruited from individuals attending the same infertility center and evaluated by the same physicians during the same time period for reasons other than infertility (e.g., routine gynecological or andrological check-ups). Fertile controls were not partners of infertile individuals.The mean age of participants in both groups was 31.4 years (range: 20–40 years). Inclusion criteria for all participants included no antibiotic use within the previous month and sexual abstinence for 4–5 days prior to semen collection. Participants who did not meet these criteria were excluded12.Demographic and clinical information—including place of residence, age, type of infertility (primary or secondary, recorded for female participants only), smoking status, and alcohol consumption—was collected using a structured questionnaire. These variables were assessed similarly in both groups to ensure comparability.Written informed consent was obtained from all participants, and all procedures were conducted in accordance with relevant guidelines and regulations, including the Declaration of Helsinki. The study protocol was approved by the Research Ethics Committee of Islamic Azad University, Kerman Branch (approval code: IR.IAU.KERMAN.REC.1401.077).The sample size was estimated based on previously reported prevalence of genital Mycoplasma infections and in consultation with a statistician to ensure adequate power for detecting relevant associations12.Sample collection and transportEndocervical samples were collected using sterile Dacron swabs and immediately transferred under aseptic conditions into 5 mL of PPLO broth. Only Dacron or polyester swabs with aluminum or plastic shafts were used, as cotton or wooden swabs may inhibit microbial growth13.Semen samples were collected after 4–5 days of sexual abstinence by masturbation into sterile containers and after 30 minutes, transferred to the laboratory of the Infertility Center. Semen quality was evaluated according to WHO guidelines, including assessment of volume, pH, motility, concentration, and morphology. These parameters were measured to ensure sample adequacy and quality; they were not included in the statistical analyses for associations with Mycoplasma infections. All procedures, including handling, transport, and disposal of samples, were conducted in accordance with institutional ethical standards.Enrichment and isolation of bacteriaFor the enrichment of M. hominis and M. genitalium, PPLO broth was prepared with 10% arginine, 10% glucose, and 5% horse serum. Penicillin G (1000 IU/mL) and polymyxin B (500 IU/mL) were added to inhibit non-target bacteria, and the final pH was adjusted to 7.0. The prepared media were transported at 4°C to the Infertility Center14.All 400 clinical samples—200 endocervical swabs and 200 semen samples—were initially inoculated into PPLO broth and incubated under 5–10% CO₂ at 37°C for 24 hours. Samples were then filtered through 0.45 µm PVDF syringe filters into fresh PPLO broth supplemented with 20% horse serum and 0.02% phenol red, followed by further incubation for 3–5 days for short-term enrichment. Bacterial growth was monitored daily based on pH-induced color changes, with yellow indicating glucose fermentation (M. genitalium) and purple indicating arginine hydrolysis (M. hominis)15.Biochemical tests—including arginine hydrolysis, glucose fermentation, urea hydrolysis, and phosphatase activity—were performed concurrently to support species identification. These biochemical results were used solely as confirmatory evidence and were not included in the final species classification, which was determined by species-specific PCR assays.Importantly, this enrichment step was not intended for quantitative or semi-quantitative culture-based prevalence assessment; rather, it was performed to preserve sample integrity and enhance the sensitivity of subsequent molecular detection. Accordingly, the prevalence reported in this study reflects PCR positivity after short-term enrichment rather than direct PCR performed on original clinical specimens. Given the slow-growing nature of genital Mycoplasma species, which typically require several weeks for detectable culture growth, the short enrichment period (3–5 days) used in this study was considered unlikely to substantially influence prevalence estimates.Following enrichment, DNA was extracted from all cultures, and PCR analysis was performed. Genus-level PCR was initially used to detect Mycoplasma spp., followed by species-specific PCR assays for M. hominis and M. genitalium. During the initial phase of the study, direct PCR was also performed on a subset of samples and yielded comparable results, supporting the reliability of the enrichment-based molecular approach.DNA extractionGenomic DNA was extracted from all enriched Mycoplasma cultures using the High Pure PCR Template Preparation Kit (Roche, Germany; Cat. No. 11796828001) following the manufacturer’s instructions.DNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Extracted DNA samples were stored at –20°C until PCR analysis.Polymerase chain reaction (PCR)PCR assays were performed to detect the Mycoplasma genus as well as M. hominis and M. genitalium species, targeting the 16S rRNA gene. Primer sequences were verified for specificity using NCBI BLAST (Table 1).Table 1 Nucleotide sequences, primers used, and their lengths.Full size tableAmplification was carried out in a T100 thermocycler (Bio-Rad, USA) using Cinagene’s (2X) Master Mix containing Taq DNA polymerase (0.5 IU/L), MgCl₂ (4 mmol/L), and dNTPs (4 mmol/L). Each reaction was prepared in a final volume of 20 μL, including 5 μL template DNA, 1 μL forward primer, 1 μL reverse primer, 3 μL deionized water, and 10 μL Master Mix (2X).The PCR thermal cycling programs were as follows:Mycoplasma genus: 35 cycles of initial denaturation at 94°C for 2 min, denaturation at 94°C for 15 s, annealing at 59.3°C for 15 s, extension at 72°C for 15 s, and a final extension at 72°C for 2 min.M. hominis: 35 cycles of initial denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 58°C for 30 s, extension at 72°C for 1 min, and a final extension at 72°C for 5 min.M. genitalium: 35 cycles of initial denaturation at 94°C for 2 min, denaturation at 94°C for 45 s, annealing at 55°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 5 min.Positive controls included the standard strain of M. hominis PG21 (ATCC: 23114)19 and M. genitalium J37 (ATCC: 33530)20, while distilled water without DNA was used as the negative control.PCR products were electrophoresed in 1.5% agarose gel, stained with Cyber Safe (CinaGen Co., Tehran, Iran), and visualized under a UV transilluminator. DNA size markers (Gene Ruler DNA Ladder Mix, Fermentas, 100–3000 bp) were used to confirm expected amplicon sizes. Bands corresponding to 272 bp, 604 bp, and 927 bp were recorded as positive for Mycoplasma genus, M. hominis, and M. genitalium, respectively, while samples without these bands were recorded as negative.Determination of minimum inhibitory concentrations (MICs) via the microdilution methodThe minimum inhibitory concentrations (MICs) of macrolides (erythromycin and azithromycin) and the fluoroquinolone (moxifloxacin) were determined for all Mycoplasma-positive samples using the 96-well microdilution method. Prior to MIC testing, samples were identified at the genus and species levels (M. hominis and M. genitalium) by PCR assays, and MIC testing was performed only on samples with confirmed molecular identification.MIC assays were conducted using enriched broth cultures derived from PCR-positive samples. In samples with single-species detection, MIC values were interpreted accordingly for the identified species. In cases of co-detection, MIC results were interpreted with caution and analyzed in the context of molecular findings.All tests were performed in duplicate to ensure reproducibility. Microplates were incubated at 37°C, and growth control wells were monitored daily for color changes over a period of 18–72 hours. The MIC was defined as the lowest antibiotic concentration that prevented visible microbial growth.Interpretive breakpoints for susceptibility (S) and resistance (R) were adopted from previously published studies specifically addressing genital Mycoplasma species, as standardized CLSI breakpoints for M. genitalium are not fully established. The applied breakpoints were as follows: erythromycin S ≤ 1 mg/L and R ≥ 4 mg/L; azithromycin S ≤ 0.125 mg/L and R ≥ 4 mg/L; and moxifloxacin S ≤ 0.25 mg/L and R ≥ 0.5 mg/L21.Investigation of antibiotic resistance mutations via sanger sequencingTo elucidate the genetic basis of antibiotic resistance, only M. genitalium isolates that demonstrated resistance to azithromycin, erythromycin, or moxifloxacin in the MIC assays were selected for sequencing. This selection strategy aligned with the study’s objective of characterizing genetic determinants specifically associated with resistant phenotypes. M. hominis isolates were not included because resistance-associated mutations in this species remain insufficiently defined, and prior research has predominantly focused on M. genitalium.Genomic DNA from resistant M. genitalium isolates was amplified using conventional PCR on a T100 thermocycler (Bio-Rad, USA), targeting the 23S rRNA gene (macrolide resistance) and the parC gene (fluoroquinolone resistance). PCR amplification was performed under the following conditions: 35 cycles consisting of initial denaturation at 98°C for 30 s; denaturation at 98°C for 10 s; annealing at 60°C for 10 s; extension at 72°C for 30 s; and a final extension at 72°C for 5 min. Primer sequences and target regions are listed in Table 2.Table 2 Nucleotide sequences, primers used, and their lengths.Full size tableAmplified products were submitted to Pishgam Company (Tehran, Iran) for bidirectional Sanger sequencing, enabling high-resolution detection of point mutations without the need for real-time PCR probe-based assays. Sequencing chromatograms were analyzed using Sequencer software (version 5.4), with particular attention to established resistance-associated loci in the 23S rRNA gene (A2058C, A2058G, A2058T, A2059C, A2059G, A2059T, A2060G) and the parC gene (A247C, G248T, G248A).This analytical approach ensured that the identified mutations could be accurately correlated with the antibiotic resistance phenotypes observed in the MIC assays.Statistical analysisStatistical analyses were conducted using SPSS software (version 29). Descriptive statistics, including frequencies and percentages, were used to summarize the demographic characteristics of the study population. For inferential analyses, parametric tests were applied to variables with a normal distribution, while non-parametric tests were used for non-normally distributed variables or categorical data. Odds ratios (ORs) were calculated using binary logistic models without adjustment for confounders. Due to the limited sample size and sparse data in some categories, all analyses are considered exploratory. All statistical tests were two-tailed, and a p-value < 0.05 was considered statistically significant.ResultsMolecular detection of mycoplasma speciesAll 400 clinical specimens—200 semen samples from men and 200 endocervical swabs from women—were analyzed using PCR. Amplification of the 16S rRNA gene (272 bp) confirmed the presence of the Mycoplasma genus in 88 samples (22%). Of these, 51 were obtained from males (48 infertile patients and 3 healthy controls) and 37 from females (32 infertile patients and 5 healthy controls) (Fig. 1). Representative gel electrophoresis images are provided; comparable amplification patterns were observed across all positive samples.Fig. 1Full size imageAmplification of a fragment of the 16S rRNA gene for the detection of the Mycoplasma genus. Lanes, from left to right: 100 bp DNA ladder (Fermentas), positive control (Mycoplasma hominis ATCC 23114), negative control (no template), and clinical samples numbered 1–12.Species-specific PCR further identified 39 cases of M. hominis (604 bp), including 23 males (22 infertile, 1 control) and 16 females (11 infertile, 5 controls). A total of 49 samples were positive for M. genitalium (927 bp): 29 males (27 infertile, 2 controls) and 20 females (all infertile cases). Coinfection with both species was detected in 4 samples (Figs. 2 & 3). Representative gel images are shown for each assay.Fig. 2Full size imageAmplification of a fragment of the 16S rRNA gene for the detection of Mycoplasma hominis. Lanes, from left to right: 100 bp DNA ladder (Fermentas), positive control (M. hominis ATCC 23114), negative control (no template), and clinical samples numbered 1–12.Fig. 3Full size imageAmplification of a fragment of the 16S rRNA gene for the detection of Mycoplasma genitalium. Lanes, from left to right: 100 bp DNA ladder (Fermentas), positive control (M. genitalium ATCC 33530), negative control (no template), and clinical samples numbered 1–12.This combined molecular workflow enabled precise detection of Mycoplasma infections at both the genus and species levels across all clinical samples.Determination of minimum inhibitory concentrations (MICs)Minimum inhibitory concentration (MIC) testing was performed on all 88 PCR-confirmed Mycoplasma-positive isolates, and MIC values were interpreted according to CLSI breakpoints for erythromycin, azithromycin, and moxifloxacin. Among the M. genitalium isolates, 14 (31.1%) demonstrated macrolide resistance, with elevated MICs for both azithromycin and erythromycin (isolates 3, 10, 20, 21, 25, 29, 44, 49, 55, 57, 62, 68, 77, and 82). Resistance to moxifloxacin was detected in two M. genitalium isolates (44 and 20), whereas all remaining isolates were susceptible to this agent.For M. hominis, five isolates (5, 22, 23, 30, and 60) were resistant to azithromycin, and six (5, 22, 23, 30, 56, and 60) exhibited erythromycin resistance. Only one M. hominis isolate23 showed resistance to moxifloxacin.MIC values for all isolates are presented in Table 3. Overall, macrolide resistance was more common in M. genitalium than in M. hominis, while resistance to moxifloxacin remained uncommon in both species.Table 3 MIC values (µg/mL) for M. genitalium and M. hominis isolates against azithromycin, erythromycin, and moxifloxacin.Full size tableDetection of antibiotic resistance-associated mutations in mycoplasma genitaliumSequencing of M. genitalium isolates that exhibited resistance to azithromycin, erythromycin, or moxifloxacin in the MIC assays revealed that the majority of resistant isolates harbored well-characterized point mutations. Specifically, 12 of 14 macrolide-resistant isolates (85.7%) carried mutations in the 23S rRNA gene: A2059G in eight isolates (isolates 3, 10, 20, 21, 25, 29, 57, 62) and A2058G in four isolates (isolates 44, 49, 55, 77). Two macrolide-resistant isolates (68 and 82) did not exhibit mutations in the sequenced regions of 23S rRNA, suggesting the possible involvement of alternative resistance mechanisms outside the analyzed loci.Among the two isolates resistant to moxifloxacin (isolates 44 and 20), sequencing of the parC gene revealed the G248T mutation in both, consistent with the observed fluoroquinolone-resistant phenotype.These findings confirm that known point mutations in the 23S rRNA and parC genes account for most of the observed resistance in M. genitalium, while also indicating that additional mechanisms may contribute to resistance in a subset of isolates. This integrated phenotypic-genotypic approach underscores the utility of targeted Sanger sequencing for identifying resistance-associated mutations in clinical isolates.Demographic and clinical profiles of infertile and control groupsThe median ages of control and infertile participants were similar for both women (29 vs. 28 years, IQR: 6–7; P = 0.790) and men (29 vs. 28 years, IQR: 6–7; P = 0.949). Among infertile women, 89% had primary infertility and 11% had secondary infertility. Lifestyle factors, including smoking and alcohol use, were not significantly associated with infertility in women (P = 0.268), whereas in men, these factors were associated with higher odds of infertility (unadjusted OR = 2.42, 95% CI: 1.21–4.87, P = 0.011).Detection of Mycoplasma genus DNA was significantly associated with infertility in both sexes. In women, infection was associated with increased odds of infertility (unadjusted OR = 8.53, 95% CI: 3.23–23.52, P < 0.001), with M. genitalium showing the strongest observed association (unadjusted OR = 48.88, 95% CI: 3.80–98.45, P < 0.001), while M. hominis was not significantly associated (P = 0.179). In men, Mycoplasma infection was associated with infertility (unadjusted OR = 31.06, 95% CI: 9.22–104.56, P < 0.001), with both M. genitalium (unadjusted OR = 19.06, 95% CI: 4.27–80.57) and M. hominis (unadjusted OR = 29.57, 95% CI: 3.90–223.09) showing significant observed associations. The extremely high odds ratios observed for some infections are likely influenced by the small number of positive cases (sparse-data effect), and results should be interpreted cautiously.Antibiotic resistance-associated mutations were detected in 8% of infertile women compared to none in controls (P = 0.007), including A2058G (2%) and A2059G (4%) mutations in the 23S rRNA gene and parC mutations (G248T) in 1% of cases. In men, resistance mutations were identified in 5% of infertile participants and 1% of controls, without a statistically significant association (P = 0.097).All odds ratios reported in this study represent unadjusted associations derived from bivariate analyses. Detailed frequencies, odds ratios, confidence intervals, and P-values are summarized in Tables 4 and 5. It should be noted that other sexually transmitted infections were not evaluated in this study; therefore, residual confounding cannot be excluded, and the observed associations should be interpreted as exploratory rather than evidence of causality.Table 4 Correlation and frequency distributions of variables in infertile and control women.Full size tableTable 5 Correlation and frequency distributions of variables in infertile and control men.Full size tableDiscussionInfertility represents a significant global health concern, and genital Mycoplasma species—particularly Mycoplasma hominis and Mycoplasma genitalium—have been suggested as potential contributors to reproductive complications, although their exact role remains incompletely understood.In this study, Mycoplasma DNA was detected in 22% of participants using PCR, including M. hominis and M. genitalium, both of which were more prevalent among infertile individuals, indicating an association rather than a causal relationship. The observed prevalence levels differed from previous reports, likely due to regional characteristics of Hormozgan Province, Bandar Abbas, including variations in sexual behaviors, access to healthcare, and environmental factors.International studies show substantial variation in M. genitalium prevalence among infertile men and women. For example, in Iran, Moosavian et al. (2019) reported a prevalence of 22% for M. hominis in infertile men and 8% in infertile women, emphasizing that PCR provides a sensitive method for detecting genital Mycoplasma species and is useful for epidemiological studies of these microorganisms. These bacteria were widespread among infertile couples in Ahvaz (southwestern Iran)11, partially consistent with our findings. Heidari Pebdeni et al. (2021) reported that genital mycoplasmas and ureaplasmas are widely present in infertile men in Kerman, Iran, and identified significant associations between M. genitalium infection and decreased sperm motility (OR = 8.06, p < 0.001)23, supporting the associative relationship observed in our male infertility cohort.In Northern Europe, Tjagur et al. (2021) reported a prevalence of 1.1% for M. genitalium and less than 2% for M. hominis using PCR, concluding that the impact of these atypical bacteria on male fertility cannot be ruled out24. These values are considerably lower than those found in our study, possibly due to population-based or regional differences Al-Jebouri et al. (2021), using conventional culture methods, reported a prevalence of 12.9% for M. hominis in infertile men in Iraq, which aligns with the generally higher prevalence observed in Middle Eastern populations25. Similarly, Le et al. (2021) in Vietnam reported a low prevalence of M. genitalium (0.79%) among infertile men using PCR, although colonization was associated with reduced sperm motility12.A more recent investigation by Abdo et al. (2024) in the United Arab Emirates assessed 308 individuals seeking fertility treatment and found a high seroprevalence of M. hominis (49%), with 23% showing evidence of ongoing infection (IgM positivity) and 9.2% indicating recent infection (IgA positivity)26. These findings highlight the associative role of M. hominis as a genital pathogen contributing to infertility, reinforcing the importance of targeted screening and awareness programs.In females, Ezeanya-Bakpa et al. (2021), using 16S rRNA gene sequencing of endocervical samples, reported that M. hominis was more prevalent than Ureaplasma urealyticum and was associated with infertilit27. Similarly, Cutoiu and Boda (2023) in Romania, using PCR, found U. urealyticum to be the most frequent genital pathogen, with some cases exhibiting M. hominis co-infection28.Regarding antibiotic susceptibility, our findings showed high levels of macrolide resistance among M. genitalium isolates, and some isolates were resistant to moxifloxacin. Comparison with Boujemaa et al. (2020), who assessed antibiotic susceptibility of M. hominis using broth microdilution, indicated that M. hominis remains highly susceptible to fluoroquinolones and doxycycline, consistent with our observations21. MIC testing followed well-established breakpoints reported in the literature, ensuring clinical and scientific validity, despite the limited standardized CLSI breakpoints for M. genitalium.In the antibiotic resistance and mutation analysis, sequencing of 23S rRNA and parC genes in resistant M. genitalium isolates revealed mutations associated with macrolide resistance (A2058G, A2059G) and fluoroquinolone resistance (G248T in parC), consistent with findings by Hilmarsdóttir et al. (2020) and Nemirosky et al. (2021)9,29. Only resistant isolates were sequenced, limiting the ability to detect silent or rare mutations in susceptible strains. Future studies should include a subset of susceptible isolates for comprehensive resistance profiling.The mechanisms by which M. hominis and M. genitalium contribute to male infertility include increased sperm apoptosis, reduced motility, and altered morphology30,31,32. In females, these microorganisms can induce endometrial inflammation, implantation failure, and an increased risk of uterine infections33,34. These findings underscore the critical role of molecular diagnostic tools. Notably, all primers used in this study were BLASTed, confirming specificity for the target species and expected amplicon size, as detailed in the Methods section.Limitations of this study include a relatively small sample size, absence of longitudinal follow-up, and lack of assessment for other sexually transmitted infections such as Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis. Consequently, residual confounding cannot be excluded, and the observed associations cannot be attributed exclusively to Mycoplasma infections. Additionally, mutation analysis was limited to the 23S rRNA and parC genes of Mycoplasma genitalium, and other resistance-associated genes, including gyrA, were not investigated due to time and financial constraints. Future studies with larger cohorts, broader pathogen screening, and mechanistic analyses are needed to clarify causal pathways and optimize therapeutic strategies.ConclusionThe findings suggest that infections with M. hominis and M. genitalium may play an important role in reproductive health and highlight the potential significance of these pathogens—alongside other sexually transmitted microorganisms—in the context of infertility. The study underscores the value of molecular diagnostics for accurate detection and the importance of monitoring emerging antibiotic resistance. These insights can inform future research and targeted screening strategies, while acknowledging that causal relationships cannot be established from a cross-sectional study design.Data availabilityThe datasets generated and/or analyzed during the current study are not publicly available due to participant privacy but are available from the corresponding author upon reasonable request.ReferencesPaira, D. A., Molina, G., Tissera, A. D., Olivera, C., Molina, R. I., Motrich, R. D. Results from a large cross-sectional study assessing Chlamydia trachomatis, Ureaplasma spp. and Mycoplasma hominis urogenital infections in patients with primary infertility. Sci. Rep. 11(1), 13655. https://www.nature.com/articles/s41598-021-93318-1 (2021).Doroftei, B., Ilie, O. D., Armeanu, T., Anton, E., Scripcariu, I., Maftei, R. The prevalence of Ureaplasma urealyticum and Mycoplasma hominis infections in infertile patients in the northeast region of Romania. Medicina (B Aires) 57(3), 211. https://www.mdpi.com/1648-9144/57/3/211 (2021).Rekha, S., Nooren, M., Kalyan, S., Mohan, M., Bharti, M., Monika, R. et al. Occurrence of Mycoplasma genitalium in the peritoneal fluid of fertile and infertile women with detailed analysis among infertile women. Microb Pathog. 129, 183–6. https://linkinghub.elsevier.com/retrieve/pii/S0882401018305035 (2019).Oh, E. J., Jang, T. S., Kim, J. K. Mycoplasma genitalium and Mycoplasma hominis infection in South Korea during 2018–2020. Iran J Microbiol. https://publish.kne-publishing.com/index.php/IJM/article/view/7423. (2021).Ozturk, S., Yildiz, S., Dursun, P., Yener Ilce, B., Kaymaz, O. Mycoplasma hominis profile in women: culture, kit, molecular diagnosis, antimicrobial resistance, and treatment. Microb Pathog. 135, 103635. https://linkinghub.elsevier.com/retrieve/pii/S0882401019300634 (2019).Mohseni Moghadam, N., Kheirkhah, B., Mirshekari, T. R., Fasihi Harandi, M., Tafsiri, E. Isolation and molecular identification of Mycoplasma genitalium from the secretion of genital tract in infertile male and female. IJRM 12(9) 601 EP. http://ijrm.ir/article-1-583-en.html (2014).Ahmadi, M.H. Resistance to tetracyclines among clinical isolates of Mycoplasma hominis and Ureaplasma species: a systematic review and meta-analysis. J Antimicrob Chemother. 76(4), 865–75. https://academic.oup.com/jac/article/76/4/865/6046148 (2021).Tsalidou, M., Stergiopoulou, T., Bostanitis, I., Nikaki, C., Skoumpa, K., Koutsoukou, T. et al. Surveillance of antimicrobial resistance and multidrug resistance prevalence of clinical isolates in a regional hospital in Northern Greece. Antibiotics 12(11), 1595. https://www.mdpi.com/2079-6382/12/11/1595 (2023).Hilmarsdóttir, I. et al. Prevalence of Mycoplasma genitalium and antibiotic resistance-associated mutations in patients at a sexually transmitted infection clinic in Iceland, and comparison of the S-DiaMGTV and Aptima Mycoplasma genitalium assays for diagnosis. J. Clin. Microbiol. https://doi.org/10.1128/JCM.01084-20 (2020).Article PubMed PubMed Central Google Scholar Zhao, L., Liu, A., Li, R. & Zhao, S. Antimicrobial resistance, genetic characterization, and molecular epidemiology of Ureaplasma species in males with infertility. Eur. J. Clin. Microbiol. Infect. Dis. 39(11), 2177–2183. https://doi.org/10.1007/s10096-020-03969-7 (2020).Article CAS PubMed Google Scholar Moosavian, M., Ghadiri, A., Amirzadeh, S., Rashno, M., Afzali, M., Ahmadi, K. Investigating Chlamydia trachomatis and genital Mycoplasma prevalence and apoptosis markers in infertile and fertile couples. Jundishapur J. Microbiol. https://brieflands.com/journals/jjm/articles/84954 (2019).Tam Le, M., Nguyen Nguyen, D., Bach Nguyen, H., Quynh Tram Ngo, V., Quoc Huy Nguyen, V. Ureaplasma urealyticum and Mycoplasma genitalium detection and sperm quality: a cross-sectional study in Vietnam. Int. J. Reprod. Biomed. 20(3), 185–94. https://knepublishing.com/index.php/ijrm/article/view/10710 (2022).Sethi, S., Zaman, K., Jain, N. Mycoplasma genitalium infections: current treatment options and resistance issues. Infect. Drug Resist. 10, 283–92. http://www.ncbi.nlm.nih.gov/pubmed/28979150 (2017).Moridi, K., Ghazvini, K., Hemmaty, M., Hoseiniun, H., Torkaman, M., Fallah Mehrabadi, M. H. Prevalence determination of M. hominis and M. genitalium in semen samples in the northeast of Iran using culture and multiplex polymerase chain reaction. Arch. Razi. Inst. 76(1), 41–9. http://www.ncbi.nlm.nih.gov/pubmed/33818956 (2021).Nazarzadeh, F., Ahmadi, M. H., Ansaripour, S., Niakan, M., Pouladi, I. Detection and evaluation of macrolide resistance (erythromycin) in Mycoplasma hominis isolated from endocervical specimens of patients referring to Ibn Sina Infertility Treatment Centre, Tehran, Iran. Int J Fertil Steril. 16(2), 95–101. http://www.ncbi.nlm.nih.gov/pubmed/35639656 (2022).Esmaeili, H., Shakeri, A. P., Tajik, P., Hamedi, M. The frequency of bacterial pathogens of mastitis and their antibiotic susceptibility in Saanen and Alpine goats. J. Med. Bacteriol. https://publish.kne-publishing.com/index.php/JMB/article/view/14371 (2023).Aguilera-Arreola, M. G., González-Cardel, A. M., Tenorio, A. M., Curiel-Quesada, E. & Castro-Escarpulli, G. Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. BMC Res. Notes 7(1), 433. https://doi.org/10.1186/1756-0500-7-433 (2014).Article CAS PubMed PubMed Central Google Scholar Matsuoka, M. et al. Characterization and molecular analysis of macrolide-resistant Mycoplasma pneumoniae clinical isolates obtained in Japan. Antimicrob. Agents Chemother. 48(12), 4624–4630. https://doi.org/10.1128/AAC.48.12.4624-4630.2004 (2004).Article CAS PubMed PubMed Central Google Scholar Molla Kazemiha, V. et al. Sensitivity of biochemical tests in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran. Cytotechnology 66(5), 861–873. https://doi.org/10.1007/s10616-013-9640-9 (2014).Article CAS PubMed PubMed Central Google Scholar Mousavi, A., Farhadifar, F., Mirnejad, R., Ramazanzadeh, R. Detection of genital mycoplasmal infections among infertile females by multiplex PCR. Iran J. Microbiol. 6(6), 398–403. http://www.ncbi.nlm.nih.gov/pubmed/25926957 (2014).Boujemaa, S., Mlik, B., Mardassi, H., Ben Abdelmoumen Mardassi B. Clonal spread of tetracycline resistance among Mycoplasma hominis clinical strains, Tunisia. Infect. Drug Resist. 2093–7. https://www.dovepress.com/clonal-spread-of-tetracycline-resistance-among-mycoplasma-hominis-clin-peer-reviewed-article-IDR (2020).He, W. et al. Detection of macrolide and fluoroquinolone resistance-associated 23S rRNA and parC mutations in Mycoplasma genitalium by nested real-time PCR. Front. Cell Infect. Microbiol. https://doi.org/10.3389/fcimb.2023.1271392/full (2023).Article PubMed PubMed Central Google Scholar Heidari Pebdeni, P., Saffari, F., Reza Mirshekari, T., Ashourzadeh, S., Taheri Soodejani, M., Ahmadrajabi, R. Bacteriospermia and its association with seminal fluid parameters and infertility in infertile men, Kerman, Iran: a cross-sectional study. Int J Reprod Biomed. 20(3), 203–12. https://knepublishing.com/index.php/ijrm/article/view/10712 (2022).Tjagur, S., Mändar, R., Poolamets, O., Pomm, K., Punab, M. Mycoplasma genitalium provokes seminal inflammation among infertile males. Int J Mol Sci. 22(24), 13467. https://www.mdpi.com/1422-0067/22/24/13467 (2021).Al-Jebouri, M. M. & Mohamed, A. A. A study on infertility of males infected with Mycoplasma hominis with reference to sperm morphology. Open J. Pathol. 11(01), 7–21. https://doi.org/10.4236/ojpathology.2021.111002 (2021).Article CAS Google Scholar Abdo NM, Aslam I, Irfan S, George JA, Alsuwaidi AR, Ahmed LA, et al. Seroepidemiology of Treponema pallidum, Mycoplasma hominis, and Ureaplasma urealyticum in fertility treatment-seeking patients in the Emirate of Abu Dhabi, United Arab Emirates. J. Infect. Public Health 17(1), 163–71. https://linkinghub.elsevier.com/retrieve/pii/S1876034123004148 (2024).Charity Ezeanya-Bakpa, C., Regina Agbakoba, N., Blanche Oguejiofor, C., Bessie Enweani-Nwokelo, I. Sequence analysis reveals asymptomatic infection with Mycoplasma hominis and Ureaplasma urealyticum possibly leads to infertility in females: a cross-sectional study. Int. J. Reprod. Biomed. 19(11), 951–8. https://knepublishing.com/index.php/ijrm/article/view/9910 (2021).Cutoiu, A. & Boda, D. Prevalence of Ureaplasma urealyticum, Mycoplasma hominis and Chlamydia trachomatis in symptomatic and asymptomatic patients. Biomed. Rep. 19(4), 74. https://doi.org/10.3892/br.2023.1656 (2023).Article CAS PubMed PubMed Central Google Scholar Lucena Nemirosky, J., Espelt, R., López Grado, E., Sobrino, J., Acera, A., Pérez, J. et al. Macrolide resistance in Mycoplasma genitalium in Catalonia, Spain: a 1 year prospective study. J. Antimicrob. Chemother. 76(10), 2702–7. https://academic.oup.com/jac/article/76/10/2702/6323546 (2021).Cheng, C. et al. Genital mycoplasma infection: a systematic review and meta-analysis. Reprod. Health 20(1), 136. https://doi.org/10.1186/s12978-023-01684-y (2023).Article PubMed PubMed Central Google Scholar Chen, J. et al. Microbiology and immune mechanisms associated with male infertility. Front. Immunol. https://doi.org/10.3389/fimmu.2023.1139450 (2023).Article PubMed PubMed Central Google Scholar Malefo, M. A., Ayo-Yusuf, O., Mokgatle, M. M. Risk factors for sexually transmitted infections among men who have sex with men. African J. Prim. Heal Care Fam. Med. 15(1). http://www.phcfm.org/index.php/PHCFM/article/view/4080 (2023).Cutoiu, A. & Boda, D. An overview regarding the relationship between Mollicutes, infertility and antibiotic resistance (Review). Biomed. Rep. 21(2), 119. https://doi.org/10.3892/br.2024.1807 (2024).Article CAS PubMed PubMed Central Google Scholar Yu, J. et al. Mycoplasma genitalium infection in the female reproductive system: Diseases and treatment. Front. Microbiol. https://doi.org/10.3389/fmicb.2023.1098276 (2023).Article PubMed PubMed Central Google Scholar Download referencesAcknowledgmentsWe acknowledge the Obstetrics Department at Ome Leila Hospital, Bandar Abbas, Iran, for facilitating patient recruitment and sample collection. Special thanks to Dr. Zahra Khashavi for her assistance and guidance during the clinical aspects of this study.Author informationAuthors and AffiliationsDepartment of Microbiology, Ke.C Islamic Azad University, Kerman, IranNeda Fazeli & Nadia KazemipourDepartment of Microbiology, Baf.C., Islamic Azad University, Baft Branch, Baft, IranBabak KheirkhahAuthorsNeda FazeliView author publicationsSearch author on:PubMed Google ScholarBabak KheirkhahView author publicationsSearch author on:PubMed Google ScholarNadia KazemipourView author publicationsSearch author on:PubMed Google ScholarContributionsNeda Fazeli: study design, data collection and analysis, manuscript drafting. Babak Kheirkhah: supervision, study design input, result interpretation, manuscript revision. Nadia Kazemipour: scientific consultation, data interpretation, manuscript revision. All authors approved the final manuscript and are accountable for all aspects of the work.Corresponding authorCorrespondence to Babak Kheirkhah.Ethics declarationsEthicsThis study was approved by the Research Ethics Committee of Islamic Azad University – Kerman Branch (Approval Code: IR.IAU.KERMAN.REC.1401.077). Informed consent was obtained from all participants.Competing interestsThe authors declare no competing interests.Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Rights and permissionsOpen Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.Reprints and permissionsAbout this article